Bacteria are particularly susceptible to acute environmental changes which require rapid adaptation for survival. These environmental changes include nutritional deficiencies, exposure to a chemical toxin, and changes in osmolarity. In order to cope with such environmental stresses, bacteria have developed a sophisticated signaling system which enables the cell to respond swiftly to any given environmental alteration. The most common signaling system in bacteria is the histidyl-aspartyl (His-Asp) phosphorelay signal transduction system. Recently His-Asp phosphorelay systems also have been identified in eukaryotic cells [Egger et al, Genes to Cells, 2:167-184 (1997); Appleby et al., Cell, 86:845-848 (1996); Inouye, Cell, 85:13-14 (1996); Parkinson and Kofoid, Ann. Rev. Gen., 26:71-112 (1992); Stock et al., Microbiol Rev., 53:450-490 (1989)].
There are two key participants in the His-Asp phosphorelay signal transduction system: (1) a sensor histidine kinase, which is generally a transmembrane protein; and (2) a response regulator which mediates changes in gene expression and/or cellular locomotion. The sensor histidine kinase responds to a particular environmental parameter by activating the response regulator. The activated response regulator then serves as a mediator of the signal to effect the cellular response to the environmental parameter. Thus, for each particular type of environmental challenge, a corresponding bacterial sensor histidine kinase exists that initiates the appropriate cellular response. Recently 23-28 open reading frames were identified in the Escherichia coli genome as encoding putative sensory kinases, whereas 32 open reading frames were identified as encoding putative response regulators [Mizuno, DNA Research., 4:161-168 (1997)].
The transmembrane sensor histidine kinase (TSHK) of the His-Asp phosphorelay signal transduction system contains a specific histidine that is autophosphorylated using ATP as the co-substrate. The TSHK can then transfer the phosphoryl group to a specific aspartyl residue of the response regulator. This phosphoryl transfer activates the response regulator and thereby mediates the signal. Unlike the analogous eukaryotic signal transduction pathways that employ either tyrosine (e.g., STATs) or threonine and/or serine (e.g., Smads) and in which the flow of the phosphoryl group is irreversible, the His-Asp pathway is based on a reversible phosphoryl transfer between histidine and aspartic acid residues.
Bacterial infections remain among the most common and deadly causes of human disease. For example, evidence of a virulent strain of E. coli in ground beef resulted in a recall of approximately $15 million worth of that food product. Such virulent strains can cause severe diarrhea, a condition which kills a million more people (3 million) each year worldwide than malaria. [D. Leff, BIOWORLD TODAY, 9:1,3 (1998)].
Although, there was initial optimism in the middle of this century that diseases caused by bacteria would be quickly eradicated, it has become evident that the so-called "miracle drugs" are not sufficient to accomplish this task. Indeed, antibiotic resistant pathogenic strains of bacteria have become common-place, and bacterial resistance to the new variations of these drugs appears to be outpacing the ability of scientists to develop effective chemical analogs of the existing drugs (See, Stuart B. Levy, The Challenge of Antibiotic Resistance, in Scientific American, 46-53 (March, 1998)). Therefore, new approaches to drug development are necessary to combat the ever-increasing number of antibiotic-resistant pathogens.
Classical penicillin-type antibiotics effect a single class of proteins known as autolysins. Thus, the development of new drugs which effect an alternative bacterial target protein would be desirable. Such a target protein ideally would be indispensable for bacterial survival. A protein involved in the His-Asp pathway such as a sensor histidine kinase would thus be a prime candidate for such drug development.
Therefore, there is a need to develop methods for identifying drugs that interfere with transmembrane sensor histidine kinase activity. Unfortunately, such identification has heretofore relied on serendipity and/or systematic screening of large numbers of natural and synthetic compounds. One superior method for drug screening relies on structure based rational drug design. In such cases, a three dimensional structure of the protein or peptide is determined and potential agonists and/or antagonists are designed with the aid of computer modeling [Bugg et al., Scientific American, December: 92-98 (1993); West et al., TIPS, 16:67-74 (1995); Dunbrack et al., Folding & Design, 2:27-42 (1997)]. Unfortunately, with the notable exception of certain sensors involved in chemotaxis, bacterial sensors tend to be transmembrane proteins having multiple domains and have heretofor not been amenable to three-dimensional structural analysis. This is due to the intrinsic difficulty in preparing high quality TSHK crystals required for X-ray crystallographic analysis and the fact that the multidomain TSHK is too large for NMR three-dimensional analysis. Therefore, there is essentially no detailed structural information for TSHKs.
Therefore, there is a need for obtaining a form of the transmembrane sensor histidine kinase that is amenable for NMR analysis and/or X-ray crystallographic analysis. In addition, there is a need for determining the three-dimensional structure of such a TSHK form. Furthermore, there is a need for developing procedures of structure based rational drug design using such three-dimensional information. Finally, there is a need to employ such procedures to develop new anti-bacterial drugs.
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